Ziram and Sodium NN-DimethyldithiocarbamateInhibit Ubiquitin Activation through Intracellular Metal Transportand Increased Oxidative Stress in HEK293 Cells

摘要

Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition,coincubations were performed with the copper chelator triethylenetetraminehydrochloride (TET). TET significantly protected E1 activity for bothof the dithiocarbamates and decreased the associated oxidative injuryin HEK293 cells as well as prevented dithiocarbamate-mediated lipidperoxidation assayed using an ethyl aracidonate micelle system. BecauseTET did not completely ameliorate intracellular transport of copperor zinc for ziram, TET apparently maintained E1 activity through itsability to diminish dithiocarbamate-mediated oxidative stress. Experimentsto determine the relative contribution of elevated intracellular zincand copper were performed using a metal free incubation system andshowed that increases in either metal were sufficient to inhibit E1.To evaluate the utility of the HEK293 in vitro system for screeningenvironmental agents, a series of additional pesticides and metalswas assayed, and eight agents that produced a significant decreaseand five that produced a significant increase in activated E1 wereidentified. These studies suggest that E1 is a sensitive redox sensorthat can be modulated by exposure to environmental agents and canregulate downstream cellular processes.